Potassium Functionally Replaces the Second Lysine of the KMSKS Signature Sequence in Human Tyrosyl-tRNA Synthetase

Joseph Austin,Eric A First

doi:10.1074/jbc.m201923200

Abstract

Unlike their bacterial homologues, a number of eukaryotic tyrosyl-tRNA synthetases require potassium to catalyze the aminoacylation reaction. In addition, the second lysine in the class I-specific KMSKS signature motif is absent from all known eukaryotic tyrosyl-tRNA synthetase sequences, except those of higher plants. This lysine, which is the most highly conserved residue in the class I aminoacyl-tRNA synthetase family, has been shown to interact with the pyrophosphate moiety of the ATP substrate in the Bacillus stearothermophilus tyrosyl-tRNA synthetase. Equilibrium dialysis and pre-steady-state kinetic analyses were used to determine the role that potassium plays in the tyrosine activation reaction in the human tyrosyl-tRNA synthetase and whether it can be replaced by any of the other alkali metals. Kinetic analyses indicate that potassium interacts with the pyrophosphate moiety of ATP, stabilizing the E.Tyr.ATP and E.[Tyr-ATP] complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyl-tRNA synthetase dimer by 0.7 kcal/mol. Rubidium is the only other alkali metal that can replace potassium in catalyzing tyrosine activation, although the forward rate constant is half of that observed when potassium is present. The above results are consistent with the hypothesis that potassium functionally replaces the second lysine in the KMSKS signature sequence. Possible implications of these results with respect to the design of antibiotics that target bacterial aminoacyl-tRNA synthetases are discussed.

Highlights

Aminoacyl-tRNA synthetases have gained attention recently as potential targets for antibiotics [1,2,3,4,5,6,7,8,9,10]
We address the question of how potassium promotes catalysis of the tyrosine activation reaction in human tyrosyl-tRNA synthetase
Based on the observation that potassium appears to functionally replace the most highly conserved amino acid in the class I aminoacyl-tRNA synthetase family, namely, the second lysine in the KMSKS signature sequence, we propose that the replacement of this lysine by potassium provides a selective advantage that has resulted in its presence being maintained in eukaryotic tyrosyl-tRNA synthetases

Summary

Introduction

Aminoacyl-tRNA synthetases have gained attention recently as potential targets for antibiotics [1,2,3,4,5,6,7,8,9,10]. The hypothesis that potassium stabilizes the transition state complex for the tyrosine activation reaction through interactions with the pyrophosphate moiety of MgATP is tested. This hypothesis is based on the following reasoning. Stabilization of the transition state for tyrosine activation in B. stearothermophilus tyrosyl-tRNA synthetase is largely due to the formation of interactions between the pyrophosphate moiety of MgATP and active site amino acids in the enzyme The second lysine in the KMSKS signature sequence, which is replaced by a serine in the human tyrosyl-tRNA synthetase, stabilizes the transition state for tyrosine activation by 3.0 kcal/mol in the B. stearothermophilus enzyme [14, 19, 21]. The observation that the stabilities of the transition states for tyrosine activation are virtually identical for the human and B

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